The present invention relates to separable compositions, methods, and kits for use in multiplexed assay detection of the interaction between ligands and target antiligands.
The need to determine many analytes or nucleic acid sequences (for example multiple pathogens or multiple genes or multiple genetic variants) in blood or other biological fluids has become increasingly apparent in many branches of medicine. Most multi-analyte assays, such as assays that detect multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences and some require sophisticated instrumentation. Some of the known classical methods for multianalyte assays include the following:
a. The use of two different radioisotope labels to distinguish two different analytes.
b. The use of two or more different fluorescent labels to distinguish two or more analytes.
c. The use of lanthanide chelates where both lifetime and wavelength are used to distinguish two or more analytes.
d. The use of fluorescent and chemiluminescent labels to distinguish two or more analytes.
e. The use of two different enzymes to distinguish two or more analytes.
f. The use of enzyme and acridinium esters to distinguish two or more analytes.
g. Spatial resolution of different analytes, for example on arrays, to identify and quantify multiple analytes.
h. The use of acridinium ester labels where lifetime or dioxetanone formation is used to quantify two different viral targets.
As the human genome is elucidated, there will be numerous opportunities for performing assays to determine the presence of specific sequences, distinguishing between alleles in homozygotes and heterozygotes, determining the presence of mutations, evaluating cellular expression patterns, etc. In many of these cases one will wish to determine in a single reaction, a number of different characteristics of the same sample. In many assays, there will be an interest in determining the presence of specific sequences, whether genomic, synthetic, or cDNA. These sequences may be associated particularly with genes, regulatory sequences, repeats, multimeric regions, expression patterns, and the like. There will also be an interest in determining the presence of one or more pathogens, their antibiotic resistance genes, genetic subtype and the like. The need to identify and quantify a large number of bases or sequences, potentially distributed over centimorgans of DNA, offers a major challenge. Any method should be accurate, reasonably economical in limiting the amount of reagents required and provide for a highly multiplexed assay, which allows for differentiation and quantitation of multiple genes, and/or snp determination, and/or gene expression at the RNA or protein level.
The need to study differential expression of multiple genes to determine toxicologically relevant outcomes or the need to screen transfused blood for viral contaminants with high sensitivity is clearly evident. Finally, while nucleic acid sequences provide extreme diversity for situations that may be of biological or other interest, there are other types of compounds, such as proteins in proteomics that may also offer opportunities for multiplexed determinations.
There is and will continue to be comparisons of the sequences of different individuals. It is believed that there will be about one polymorphism per 1,000 bases, so that one may anticipate that there will be an extensive number of differences between individuals. By single nucleotide polymorphism (SNPs) is intended that there will be a prevalent nucleotide at the site, with one or more of the remaining bases being present in a substantially smaller percent of the population. While other genetic markers are available, the large number of SNPs and their extensive distribution in the chromosomes make SNPs an attractive target. Also, by determining a plurality of SNPs associated with a specific phenotype, one may use the SNP pattern as an indication of the phenotype, rather than requiring a determination of the genes associated with the phenotype. For the most part, the SNPs will be in non-coding regions, primarily between genes, but will also be present in exons and introns. In addition, the great proportion of the SNPs will not affect the phenotype of the individual, but will clearly affect the genotype. The SNPs have a number of properties of interest. Since the SNPs will be inherited, individual SNPs and/or SNP patterns may be related to genetic defects, such as deletions, insertions and mutations, involving one or more bases in genes. Rather than isolating and sequencing the target gene, it will be sufficient to identify the SNPs involved. In addition, the SNPs may also be used in forensic medicine to identify individuals.
Thus an assay for the differentiation and quantitation of multiple genes, and/or snp determination, and/or gene expression at the RNA or protein level, that has higher sensitivity, a large dynamic range (103 to 104-fold differences in target levels), a greater degree of multiplexing, and fewer and more stable reagents would increase the simplicity and reliability of multianalyte assays, and reduce their costs.
Holland (Proc. Natl. Acad. Sci. USA (1991) 88:7276) discloses that the exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase in PCR amplification to generate specific detectable signal concomitantly with amplification.
The TaqMan(copyright) assay is discussed by Lee in Nucleic Acid Research (1993) 21:16 3761).
White (Trends Biotechnology (1996) 14(12):478-483) discusses the problems of multiplexing in the TaqMan assay.
Marino, Electrophoresis (1996) 17:1499 describes low-stringency-sequence specific PCR (LSSP-PCR). A PCR amplified sequence is subjected to single primer amplification under conditions of low stringency to produce a range of different length amplicons. Different patterns are obtained when there are differences in sequence. The patterns are unique to an individual and of possible value for identity testing.
Single strand conformational polymorphism (SSCP) yields similar results. In this method the PCR amplified DNA is denatured and sequence dependent conformations of the single strands are detected by their differing rates of migration during gel electrophoresis. As with LSSP-PCR above, different patterns are obtained that signal differences in sequence. However, neither LSSP-PCR nor SSCP gives specific sequence information and both depend on the questionable assumption that any base that is changed in a sequence will give rise to a conformational change that can be detected. Pastinen, Clin. Chem. (1996) 42:1391 amplifies the target DNA and immobilizes the amplicons. Multiple primers are then allowed to hybridize to sites 3xe2x80x2 and contiguous to a SNP (single nucleotide polymorphism) site of interest. Each primer has a different size that serves as a code. The hybridized primers are extended by one base using a fluorescently labeled dideoxynucleoside triphosphate. The size of each of the fluorescent products that is produced, determined by gel electrophoresis, indicates the sequence and, thus, the location of the SNP. The identity of the base at the SNP site is defined by the triphosphate that is used. A similar approach is taken by Haff, Nucleic Acids Res. (1997) 25:3749 except that the sizing is carried out by mass spectrometry and thus avoids the need for a label. However, both methods have the serious limitation that screening for a large number of sites will require large, very pure primers that can have troublesome secondary structures and be very expensive to synthesize.
Hacia, Nat. Genet. (1996) 14:441 uses a high-density array of oligonucleotides. Labeled DNA samples were allowed to bind to 96,600 20-base oligonucleotides and the binding patterns produced from different individuals were compared. The method is attractive in that SNPs can be directly identified, but the cost of the arrays is high and non-specific hybridization may confound the accuracy of the genetic information.
Fan (Oct. 6-8, 1997 IBC, Annapolis Md.) has reported results of a large scale screening of human sequence-tagged sites. The accuracy of single nucleotide polymorphism screening was determined by conventional ABI resequencing.
Ross in Anal. Chem. (1997) 69:4197 discusses allele specific oligonucleotide hybridization along with mass spectrometry.
Brenner and Lerner, PNAS (1992) 89:53 81, suggested that compounds prepared by combinatorial synthesis can each be labeled with a characteristic DNA sequence. If a given compound proves of interest, the corresponding DNA label is amplified by PCR and sequenced, thereby identifying the compound.
W. Clark Still, in U.S. Pat. No. 5,565,324 and in Accounts of Chem. Res., (1996) 29:155, uses a releasable mixture of halocarbons on beads to code for a specific compound on the bead that is produced during synthesis of a combinatorial library. Beads bearing a compound of interest are treated to release the coding molecules and the mixture is analyzed by gas chromatography with flame ionization detection.
U.S. Pat. No. 5,807,682 describes probe compositions for detecting a plurality of nucleic acid targets.
Methods and compounds are provided for multiplexed determinations, where the compounds can be linked to binding compounds for detection of reciprocal binding compounds in a sample. The methods are distinguished by having a plurality of binding events in a single vessel using a mixture of differentially eTag reporter-conjugated binding compounds, the release of identifying eTag reporters of those binding compounds bound to their target compounds in the same vessel, and the detection of the released identifying tags by separation of the tags in a single run. The eTag reporters are distinguished by having one or more physical characteristics that allow them to be separated and detected.
The method employs a mixture of binding compounds bound to eTag reporters, where each eTag reporter has a characteristic that allows it to be uniquely detected in a single separation run. The method involves combining the etag reporter conjugated binding compound with a sample to determine the presence of a plurality of targets under conditions where the binding compounds bind to any reciprocal binding partners to form a binding complex. After sufficient time for binding to occur, the eTag reporters can be released from binding complexes in the same vessel. Various techniques are employed depending upon the nature of the binding compounds for releasing the eTag reporters bound to the complex. The released eTag reporters are then separated and identified by their differentiable characteristics free of interference from the eTag reporters still bound to the binding compound. The techniques for differentiating between eTag reporters bound to a complex and not bound to a complex, include enzymatic reactions that require the complex to exist for cleavage to occur, modification by using ligand/receptor binding, where the ligand is part of the binding compound, so that after cleavage, eTag reporter still bound to the binding compound is modified, dual binding to the target resulting in release of the eTag receptor, where optionally eTag reporter bound to the binding compound is modified, and the like.
One set of eTag reporters are distinguished by differences, which include mass as a characteristic. These eTag reporters do not rely on differentiation based on oligonucleotides of 2 or more, usually 3 or more nucleotides, but rather on organic chemical building blocks that are conveniently combined together to provide for large numbers of differentiable compounds. Therefore, while the original eTag reporter or eTag reporter conjugated to the binding compound can have 2 or more nucleotides, when released from the binding compound, the released eTag reporter will have not more than 3, usually not more than 2 nucleotides. Of particular interest are eTag reporters that are characterized by differences in their mass/charge ratio. These compounds are distinguished by having differences in mobility and are characterized by having regions, which serve as (1) a cleavable linking region; (2) a mass-modifying region; (3) a charge-modifying region: and (4) a detectable region, where the regions may be separate and distinct or combined, there being at least two distinct regions that provide for the differentiation. These eTag reporters may be combined in kits and assays with compounds having all of the regions within a single region to further expand the number of different compounds used as eTag reporters in a multiplexed determination. These compounds find use with other compounds where the different regions are present in the same moiety, for example one to two regions, where the charge-modifying region may also be the detectable region or the mass-modifying region. By having a plurality of compounds that can serve as identifying molecules, mixtures of target compounds can be assayed in a single vessel. By using protocols that result in the release of eTag(trademark) reporters from the binding compound that are identifiable due to differences in mobility, the analysis is greatly simplified, since the eTag reporters will be substantially free of interfering materials and their differences in mobility will allow for accurate detection and quantitation.
Sets of electrophoretic tag (e-tag) probes for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided.
The e-tag probe sets comprise j members, and have the form: (D, Mj)-L-Tj, where (a) D is a detection group comprising a detectable label; (b) Mj is a mobility modifier, having a particular charge/mass ratio; (c) Tj is a ligand capable of binding to or interacting with a target antiligand and (d) L is a linking group connected to Tj by a bond that is cleavable by a selected cleaving agent when the probe is bound to, or interacting with, the target antiligand.
When the ligand of an e-tag probe of the set interacts with a target antiligand and is treated with a selected cleaving agent, a mixture of e-tag reporters having the form (D, Mj)-Lxe2x80x2, and uncleaved and/or partially cleaved probes is produced. Lxe2x80x2 is the residue of L attached to (D, Mj) after such cleavage.
The mobility modifier imparts a unique and known electrophoretic mobility to each released e-tag reporter which is within a selected range of electrophoretic mobilities with respect to other e-tag reporters of the same form in the probe set.
The sets include e-tag probes having the form, Mj-D-L-Tj and D-Mj-L-Tj, with the corresponding e-tag reporters having the form Mj-D-Lxe2x80x2 and D-Mj-Lxe2x80x2, respectively.
Uncleaved or partially cleaved e-tag probes, but not the corresponding e-tag reporter, have one or more chemical groups capable of reacting with or binding to a selected capture agent. The capture either (i) imparts a mobility to probes bound to the capture agent that prevent the probes from electrophoretically migrating within a selected range of electrophoretic mobilities or (ii) immobilizes the probes on a solid support.
In one aspect, the target is a nucleotide, and Tj is an oligonucleotide target-binding moiety having a sequence of nucleotides Ui connected by intersubunit linkages Bi, i+1, where i includes all integers from 1 to n, and n is sufficient to allow the target-binding moiety to hybridize specifically with a target nucleotide sequence.
In this aspect, L is a nucleotide joined to U1 in Tj through a nuclease-cleavable bond; and target-binding moiety of each e-tag probe of the set contains at least one modification selected from the following:
(i) at least one nuclease-resistant bond Bi, i+1, where i includes at least 1;
(ii) a capture ligand, e.g., biotin, bound to U1 and capable of binding specifically to a capture agent; and
(iii) a nuclease-resistant bond Bi, i+1, where i includes at least 1, and a capture ligand bound to at least one nucleotide Ui and capable of binding specifically to a capture agent, where ixe2x89xa71.
Exemplary probes sets include those: (i) where the linking group includes at least a portion of an amino acid sequence that is recognized and cleaved by a selected peptidase; (ii) where the linking group includes at least a portion of an oligosaccharide that is recognized and cleaved by a selected hydrolytic enzyme; (iii) where L and Tj are linked by an ester linkage that is cleaved by a selected esterase; (iv) where L and Tj are linked by a disulfide bond, and the antiligand is attached to an oxidase enzyme, such that in the presence of a substrate for the enzyme, H2O2 generated by the oxidase is effective to cleave the disulfide linkage in a probe bound to the antiligand; (v) where L and Tj are linked by a bond cleavable by singlet oxygen, wherein the antiligand is attached to a sensitizer capable of generating singlet oxygen when photoactivated; (vi) useful in screening lignads for the ability to bind to a receptor, wherein the ligands are represented by Tj and are members of a combinatorial library of small organic molecules, and the antiligand is the receptor; and (vii) useful in screening substrates of a selected enzyme antiligand, wherein the substrate comprises a fixed moiety L and a variable moiety Tj, and interaction of a substrate probe with the enzyme is effective to cleave the substrate to release the Tj moiety from the substrate.
In the probe sets of the invention, the mobility modifier may have one or more of the following characteristics:
(i) a unique charge/mass ratio due to variations in mass, but not charge;
(ii) a unique charge/mass ratio due to changes in both mass and charge;
(iii) a unique charge/mass ratios of between xe2x88x920.001 and 0.5;
(iv) a structure that includes a selected number of negatively charged and/or positively charged amino acids; and
(v) a structure that includes an alkyl chain, and differs from other Mj in the set by 1-3 methylene groups in the chain.
In one aspect, the probe sets comprise at least 5 probes, and in a related aspect, the probe sets comprise at least 9 probes.
The label component of the probe sets of the invention may be a fluorophore, a chromophore, or an electrochemical compound capable of a detectable reaction in the presence of a redox agent. In addition, the label component of the probe set may have a selected mass and charge.
Further, a given set of e-tags probes for multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands may contain subsets of probes, each subset having a label with a unique mass/charge ratio.